The present invention relates generally to the field of cell physiology, and more particularly, to programmed cell death, or apoptosis. The novel peptides, compositions and methods of the invention are useful for modulating apoptosis in cells.
The phenomenon of programmed cell death, or xe2x80x9capoptosis,xe2x80x9d is involved in and important to the normal course of a wide variety of developmental processes, including immune and nervous system maturation. Apoptosis also plays a role in adult tissues having high cell turnover rates (Ellis, R. E., et al., Ann. Rev. Cell. Biol. 7: 663-698 (1991); Oppenheim, R. W., Ann. Rev. Neurosci. 14: 453-501 (1991); Cohen, J. J., et al., Ann. Rev. Immunol. 10: 267-293 (1992); Raff, M. C., Nature 356: 397-400 (1992)). In addition to its role in development, apoptosis has been implicated as an important cellular safeguard against tumorigenesis (Williams, G. T., Cell 65: 1097-1098 (1991); Lane, D. P., Nature 362: 786-787 (1993)). Under certain conditions, cells die by apoptosis in response to high-level or deregulated expression of oncogenes (Askew, D., et al., Oncogene 6: 1915-1922 (1991); Evan, G. I., et al., Cell 69: 119-128 (1992); Rao, L., et al., Proc. Natl. Acad. Sci. USA 89: 7742-7746 (1992); Smeyne, R. J., et al., Nature 363: 166-169 (1993); Tanaka, S., et al., Cell 77: 829-839 (1994); Wu, X., et al., Proc. Natl. Acad. Sci. USA 91: 3602-3606 (1994)). Suppression of the apoptotic program, by a variety of genetic lesions, may contribute to the development and progression of malignancies. This is well illustrated by the frequent mutation of the p53 tumor suppressor gene in human tumors (Levine, A. J., et al., Nature 351: 453-456 (1991)).
Other factors have been identified which appear to play roles in regulating apoptosis. One of these, the Insulin-Like Growth Factor-I Receptor (IGF-IR), is a member of the tyrosine kinase family of signal transducing molecules. The IGF-IR is activated by the ligands IGF-I, IGF-II and insulin at supra-physiological concentrations, and plays an important role in the development, growth, and survival of normal cells (LeRoith, D., et al., Endocrine Revs. 16: 143-163 (1995); Lowe, W. L., Jr. xe2x80x9cBiological actions of the Insulin-like growth factor receptors,xe2x80x9d in LeRoith, D., Ed., Insulin-like Growth Factors: Molecular and Cellular Aspects, CRC Press, Boca Raton, Pub. (1991); Baserga, R., et al., Cell Prolif. 27: 63-71 (1994)). Over-expression of the IGF-IR leads to the transformation of fibroblasts and conversely, IGF-IR null fibroblasts are refractory to transformation by a number of oncogenes (Sell, C., et al., Mol. Cell Biol. 14: 3604-3612 (1994)).
There is considerable evidence for a role for the IGF-IR in the maintenance of tumor cells in vitro and in vivo. IGF-IR levels are elevated in tumors of lung (Kaiser, U., et al., J. Cancer Res. Clin Oncol. 119: 665-668 (1993); Moody, T. W. and Cuttitta, F., Life Sciences 52: 1161-1173 (1993)), breast (Pollak, M. N., et al., Cancer Lett. 38: 223-230 (1987); Foekens, J. A., et al., Cancer Res. 49: 7002-7009 (1989) Cullen, K. I., et al., Cancer Res. 49: 7002-7009 (1990)) and colon (Remaole-Bennet, M. M., et al., J. Clin. Endocrinol. Metab. 75: 609-616 (1992); Guo, Y. S., et al., Gastroenterol. 102: 1101-1108 (1992)). Increased levels of IGF-I and/or IGF-II expression have been associated with human tumors (McCauley, V. M., et al., Cancer Res. 50: 2511-2517 (1990); Bhatavdekar, J. M., et al., Neoplasma 41: 101-103 (1994)). Many of these tumor cell types respond to IGF-I with a proliferative signal in culture (Nakanishi, Y., et al., J. Clin. Invest. 82: 354-359 (1988); Freed, K. A. and Herrington, A. C., J. Mol. Endocrinol. 3: 509-514 (1989)), and autocrine or paracrine loops for proliferation in vivo have been postulated (LeRoith, D., et al., Endocrine Revs. 16: 143-163 (1995); Yee, D., et al., Mol. Endocrinol. 3: 509-514 (1989)).
IGF-I protects from apoptosis induced by cytokine withdrawal in IL-3-dependent hemopoietic cells (Rodriguez-Tarduchy, G., et al., J. Immunol. 149: 535-540 (1992)), and from serum withdrawal in Rat-1/mycER cells (Harrington, E., et al., EMBO J. 13: 3286-3295 (1994)). Of cytokines present in fetal bovine serum, including the mitogens EGF and PDGF, IGF-I proved to be the most potent in inhibition of myc-induced death in Rat-1 cells. The anti-apoptotic function of IGF-I was evident in the post-commitment stage of the cell cycle and also in cells blocked in cell cycle progression by etoposide or thymidine.
The demonstration that c-myc driven fibroblasts are dependent on IGF-I for their survival suggests that there is an important role for the IGF-IR in the maintenance of oncogene driven tumor cells by specifically inhibiting apoptosis, a role distinct from the better characterized proliferative effects. This would be similar to a role thought to be played by other anti-apoptotic genes such as bcl-2 in promoting tumor survival (McDonnell, T. J., et al., Cell 57: 79-88 (1989); Hockenberry, D. M., et al., Nature 348: 334-336 (1990)). The protective effects of IGF-I are dependent upon receptor levels rather than on availability of the ligand (Resnicoff, M., et al., Cancer Res. 55: 3739-3741 (1995a)). Support for an anti-apoptotic function of IGF-IR in the maintenance of tumor cells was also provided by a study using antisense oligonucleotides to the IGF-IR that identified a quantitative relationship between IGF-IR levels, the extent of apoptosis and the tumorigenic potential of a rat syngeneic tumor (Rescinoff, M., et al., Cancer Res. 55: 3739-3741 (1995b)).
Fibroblasts from IGF-IR null mice have been used to demonstrate a requirement for the IGF-IR in transformation, and also to map domains in the receptor essential for the proliferative and transformation function of the IGF-IR. Specifically, the C terminal region of the IGF-IR is required for the transformation function. Receptors which are truncated at amino acid 1229 fail to transform fibroblasts derived from IGF-IR null mice, but retain full proliferative activity (Surmacz, E., et al., Exp. Cell Res. 218: 370-380 (1995)). Within the C-termninal region, the transforming activity has been further localized to a domain between amino acids 1245 and 1294; substitution of the single tyrosine 1251 with phenylalanine impairs transformation function (Miura, M., et al., J. Biol. Chem. 270: 22639-22644 (1995b)), substitution of the four serines (1280-1283) completely abolishes transformation (Li et al., submitted), and substitution of histidine 1293 and lysine 1294 reduces transformation activity (Hongo et al., submitted). All of the transformation-defective, truncated and point mutant receptors retain proliferative capacity. These studies indicate that two separate functions of the receptor, proliferation and transformation, are spatially distinct within the receptor and that transformation may need additional signals to those required for proliferation. Mutations at the ATP binding site in the kinase domain, at the tyrosine cluster in the kinase domain, or at tyrosine 950 (the major binding site for well defined substrates of the IGF-IR, IRS-1 and SHC) abolish both proliferation and transformation (Miura, M., et al., Cancer Res. 55: 663-667 (1995a); Li, S., et al., J. Biol. Chem. 269: 32558-32564 (1994); Gronberg, M., et al., J. Biol. Chem. 268: 23435-23440 (1993)).
As the preceeding discussion demonstrates, while recent studies have advanced the general understanding of the transformation and proliferative functions of the IGF-IR in vertebrate cells, the apparent anti-apoptotic function of the IGF-IR remains less well characterized. Elucidation of IGF-IR domains involved in the receptor""s anti-apoptotic function would be of great value in the development of compositions which modulate the survival of certain cells, such as cancer cells. The ability to modulate the anti-apoptotic activity of the IGF-IR would also allow the development of compositions and strategies for treating cells affected by diseases, such as neurodegenerative diseases, and by acute hypoxic injury, such in stroke, in which activation of the IGF-IR""s anti-apoptotic function would be beneficial. Conversely, inactiviation of the anti-apoptotic function of the IGF-IR in tumor cells would be a useful and specific treatment strategy. Accordingly, a need persists to identify the potential domain(s) in the IGF-IR responsible for its anti-apoptotic function.
In a broad aspect, the present invention is directed to novel compositions comprising domains of the IGF-IR that regulate the survival or anti-apoptotic function of the receptor upon ligand binding. These novel compositions, collectively referred to herein as xe2x80x9cActive Survival Domains,xe2x80x9d comprise peptide regions of the IGF-IR, modulation of which enhances or diminishes anti-apoptotic response upon receptor activation. Modulation of the Active Survival Domains of the IGF-IR, as well as compositions which effect such modulation, form additional broad aspects of the invention.
In one embodiment, the present invention is directed to C-terminal deletion mutants of the IGF-IR. Vertebrate cells comprising the C-terminal deletion mutants of the invention exhibit enhanced anti-apoptotic response upon ligand binding to the receptor. C-terminal deletion mutants according to this aspect of the invention include, but are not limited to, deletion mutants 1229d, 1245d and 1293d (i.e., mutant IGF-IR peptides truncated at amino acids F1229, R1245 and H1293, respectively), and their functional equivalents.
In another aspect, the invention is directed to isolated and purified C-terminal peptides, which may alternatively be synthetically produced, comprising the last 108, 92 or 44 amino acids of the IGF-IR, preferably comprising IGF-IR cytoplasmic domain constructs designated MyCF, CF, MyCF-N, MyCF-mid, MyCF-C, MyCF-29, MyCF-62, CF-N, CF-mid, and CF-C, or constructs of MyCF, CF and MyCF-N having mutations at Y1250F/Y1251F, H1293F/K1294R or S1280-1283A, and to molecules that mimic and/or interfere with their structure and/or function, useful for inducing or modulating the apoptotic state of a cell. Chemical compounds that disrupt the function of the Active Survival Domain have utility as apoptosis-modulating agents. Accordingly, in yet another aspect, the invention is directed to agents capable of disrupting Active Survival Domain function. Such agents include, but are not limited to, molecules that bind to the Active Survival Domain, molecules that interfere with the interaction of the Active Survival Domain with other peptide sequences derived from cellular proteins (including, but not limited to, the 108, 92 and 44 amino acid C-terminal peptides or other peptide sequences of the IGF-IR itself), and molecules comprising the Active Survival Domain which is altered in some manner.
The invention further provides screening methods to identify molecules that modulate apoptosis by disrupting the function of the Active Survival Domain by, for example, intramolecular interaction with the IGF-IR, which accordingly comprise additional contemplated embodiments. In one aspect, such screening methods comprise competitive binding assays wherein the ability of a putative modulating molecule to bind to truncated 1229d, 1254d or 1293d deletion mutants of the IGF-IR is measured in the presence of a suitably labeled C-terminal peptide.
In another embodiment, the invention is directed to single or multiple point mutants of the IGF-IR. Vertebrate cells comprising the point mutated IGF-IR of the invention exhibit altered anti-apoptotic response upon ligand binding to the receptor. Point mutated IGF-IR compositions according to this aspect of the invention include, but are not limited to, the mutant Y1251F and the double mutants Y1250F/Y1251F and H1293F/K1294R, and their functional equivalents. The phosphorylation state of the point mutated IGF-IR compositions of the invention comprises an additional aspect of the invention, as does the modulation of that state, which may be accomplished according to the invention through, for example, inhibition of the respective protein tyrosine kinase or phosphotyrosine phosphatase, by means of which the anti-apoptotic signal of the point mutated IGF-IR compositions of the invention may be affected.
Yet additional embodiments of the invention comprise the use of the point mutated IGF-IR compositions of the invention as screening markers for molecules which modulate apoptosis. Such embodiments include, but are not limited to, assays which measure the ability of a putative apoptosis modulating molecule to compete with other peptides and proiteins (including, but not limited to, other peptide sequences of the IGF-IR itself), which are identified to bind specifically to the point mutated IGF-IR compositions of the invention, in order to modulate the apoptotic state of a cell. In one specifically contemplated exemplary embodiment are provided assays in which the ability of a putative apoptosis modulating molecule to bind to a point mutated receptor is reduced or lost or gained when measured against the same molecule""s affinity for the wild type (i.e., non-mutated) receptor.
Molecules identified by means of the screening assays of the invention will be candidates as useful therapeutic drugs for the in vivo, ex vivo or in vitro treatment of target cells alone or in combination with suitable carriers and excipients. Such compositions and their use comprise additional embodiments of the invention.
In additional aspects, the present invention relates to products and processes involved in the cloning, preparation and expression of C-terminal mutant IGF-IR compositions and point mutated IGF-IR compositions according to the invention (collectively, xe2x80x9cmutant IGF-IR compositionsxe2x80x9d); antibodies with specificity to these mutant IGF-IR compositions; and nucleotide sequences encoding these mutant IGF-IR compositions or portions thereof. Peptides comprising the mutant IGF-IR compositions of the invention are useful for producing monoclonal and polyclonal antibodies thereto. Such antibodies, and fragments thereof, are useful for detecting and isolating proteins comprising the mutant IGF-IR compositions in biological specimens including, for example, cells from all human tissues including heart tissue, lung tissue, tumor cells, brain tissue, placenta, liver, skeletal muscle, kidney, and pancreas, as well as for modulating the apoptotic activity of proteins comprising the mutant IGF-IR compositions, such as C-terminal fragments, in and from such biological specimens, and constitute additional aspects of the invention.
In yet another aspect, the invention provides for expression vectors containing genetic sequences, hosts transformed with such expression vectors, and methods for producing the recombinant mutant IGF-IR compositions of the invention.
The present invention is further directed to methods for inducing or suppressing apoptosis in the cells and/or tissues of individuals suffering from disorders characterized by inappropriate cell proliferation or survival, or by inappropriate cell death, respectively. Disorders characterized by inappropriate cell proliferation and/or survival include, for example, inflammatory conditions, cancer, including lymphomas, genotypic tumors, etc. Disorders characterized by inappropriate cell death include, for example, autoimmune diseases, acquired immunodeficiency disease (AIDS), cell death due to radiation therapy or chemotherapy, acute hypoxic injury, etc.
The present invention also relates to methods for detecting the presence of the IGF-IR anti-apoptotic domain, as well as methods directed to the diagnosis of disorders, which disorders are associated with an increased or decreased level of expression of proteins comprising the IGF-IR anti-apoptotic domain, as compared to the expected level of expression of such proteins in the normal cell population.
The present invention relates to the therapeutic use of peptides comprising the IGF-IR anti-apoptotic domain.
The present invention also relates to methods for modulating the apoptotic state of a cell by administering peptides comprising Active Survival Domain sequences or compounds that modulate the activity of the Active Survival Domain to an individual suffering from a disorder characterized by inappropriate cell proliferation or inappropriate cell death, in order to stabilize inappropriate cell proliferation (i.e., induce apoptosis) or stabilize inappropriate cell death (i.e., suppress apoptosis), respectively, and/or in either case to restore normal cell behavior.
In another aspect, the present invention is directed to the surprising discovery that C-terminal amino acid peptides from the IGF-IR, including the C-terminal 108 amino acid peptide, are cytotoxic to tumor cells. These peptides specifically inhibit and/or kill cells which are dependent upon the IGF-IR C-terminus, i.e., cells which exhibit anchorage-independent growth and/or apoptotic stimulus provided by transfection in vitro or growth in a biodiffusion chamber in vivo. The cytotoxic C-terminal amino acid peptides (and their functional analogs) of the invention are useful as therapeutic agents and in screening assays for other agents which modulate the anti-apoptotic function of the IGF-IR.
In yet another aspect, the invention is directed to a method of assaying IGF-IR anti-apoptotic function in IL-3-dependent cells, which method allows the demonstration of the dissociation of survival function from mitogenic and transforming functions in the receptor structure.
These and other objects and aspects of the invention will be apparent to those of skill from the description which follows.